Mechanisms of Reduced Nitric Oxide/cGMP–Mediated Vasorelaxation in Transgenic Mice Overexpressing Endothelial Nitric Oxide Synthase

Abstract

Abstract-NO, constitutively produced by endothelial NO synthase (eNOS), plays a key regulatory role in vascular wall homeostasis. We generated transgenic (Tg) mice overexpressing eNOS in the endothelium and reported the presence of reduced NO-elicited relaxation. The purpose of this study was to clarify mechanisms of the reduced response to NO-mediated vasodilators in eNOS-Tg mice. Thoracic aortas of Tg and control mice were surgically isolated for vasomotor studies. Relaxations to acetylcholine and sodium nitroprusside were significantly reduced in Tg vessels compared with control vessels. Relaxations to atrial natriuretic peptide and 8-bromo-cGMP were also significantly reduced in Tg vessels. Reduced relaxations to these agents were restored by chronic N G -nitro-L-arginine methyl ester treatment. Basal cGMP levels of aortas were higher in Tg mice than in control mice, whereas soluble guanylate cyclase (sGC) activity in Tg vessels was Ϸ50% of the activity in control vessels. Moreover, cGMP-dependent protein kinase (PKG) protein levels and PKG enzyme activity were decreased in Tg vessels. These observations indicate that chronic overexpression of eNOS in the endothelium resulted in resistance to the NO/cGMP-mediated vasodilators and that at least 2 distinct mechanisms might be involved: one is reduced sGC activity, and the other is a decrease in PKG protein levels. We reported for the first time that increased NO release from the endothelium reduces sGC and PKG activity in mice. These data may provide a new insight into the mechanisms of nitrate tolerance and cross tolerance to nitrovasodilators. Key Words: nitric oxide synthase Ⅲ mice, transgenic Ⅲ guanylyl cyclase Ⅲ protein kinases N itric oxide plays critical roles in vascular biology, including its action on vascular tone and regulation of vascular structure. 1 In physiological conditions, NO is mainly produced by endothelial NO synthase (eNOS) in vessels. eNOS produces small amounts of NO continuously by physiological stimuli, such as shear stress and endogenous vasoactive substances. 2 Released NO diffuses to overlying vascular smooth muscle and binds to the ferrous heme moiety of soluble guanylate cyclase (sGC), thereby activating the enzyme. Activated sGC converts guanosine triphosphate to the intracellular second messenger cGMP, which relaxes vascular smooth muscle cells. 3 Most of the effects of cGMP are mediated by stimulation of the cGMP-dependent protein kinase (PKG). 4 Recently, we generated transgenic (Tg) mice overexpressing the bovine eNOS gene in endothelial cells. The Tg mice exhibited increased basal NO production and basal cGMP levels in the vascular wall. Furthermore, in previous reports, we found that the overproduction of NO caused reduced endothelium-dependent and NO-mediated relaxations without changes in cAMP-mediated relaxation. 5,6 Those were the first reports indicating that increased intrinsic NO induced resistance to NO-mediated vasodilators. This alteration in vascular reactivity resembles "nitrate tolerance." Organic nitrates i...

Enhancement of Ischemia-Induced Angiogenesis by eNOS Overexpression

“…We established mice overexpressing eNOS in the vascular endothelium exhibited a NO/cGMPdependent decrease in blood pressure, 2 whereas we also found that eNOS overexpression reduced the sGC activity and PKG expression leading to reduction of endotheliumdependent or endothelium-independent activities. 12 This raised the possibility that continuous activation of endogenous NO may impair angiogenesis. Our present study thus extended the work of Murohara et al 11 to establish that endogenous NO production enhances angiogenesis in hindlimb ischemia affecting neither ischemia-induced VEGF expression nor VEGF-mediated Akt-eNOS signaling.…”

“…2,12 The tissues remaining after cGMP measurement were digested by use of a bicinchoninic acid protein assay kit (Pierce). Plasma nitrite and nitrate (NOx) were measured by the Griess method, as previously described.…”

“…However, we have reported that NO overexpression attenuates NO/cGMP-mediated vasoactive actions by reducing soluble guanylate cyclase (sGC) activity and cGMPdependent protein kinase (PKG) levels. 12 Although NO was shown to downregulate the expression of VEGF gene, [13][14][15][16] there is a considerable body of evidence that NO upregulates VEGF gene expression in various cell types. [17][18][19] Jozkowicz et al 17 reported that NO derived from NO donors or generated by NOS within the cells upregulates the synthesis of VEGF in vascular smooth muscle cells.…”

Endothelial NO Synthase Overexpression Inhibits Lesion Formation in Mouse Model of Vascular Remodeling

“…10 We also found that the basal eNOS activity in the vessels showed a 7-fold increase and that acetylcholineinduced NO production was elevated 3-fold in eNOS-Tg mice compared wild-type mice. 10,11 In the present study, to clarify the effects of chronic NO overproduction on vascular remodeling, particularly on lesion formation, we examined, with the use of eNOS-Tg mice, the mouse carotid artery vascular remodeling model 12 in which the endothelium remains uninjured.…”

Statins prevent adverse effects of postnatal glucocorticoid therapy on the developing brain in rats

“…It is likely that the fall in plasma NOx following statin treatment alone is the result of negative feedback due to excess NO bioavailability and thereby having no adverse effects on the brain. Chronic increases in NO have been reported to down-regulate sGC expression (33) and to increase phosphodiesterase function (34), lowering the bioavailability of NO. This idea is consistent with dexamethasone-induced reductions in the concentration of the vasodilator gas with deleterious effects on the brain, vs. effects of statins in dexamethasone-treated pups replenishing appropriate NO concentrations and therefore, protecting the brain.…”